摘要
目的观察慢性缺氧对小鼠心肌细胞自噬水平的影响,并初步探讨AMPKα2敲除对该过程的作用。方法采用野生型与AMPKα2^(-/-)雄性C57小鼠均分为野生型常氧组、野生型缺氧组、敲除型常氧组和敲除型缺氧组(n=10)。常氧组于空气环境中饲养,缺氧组于10%O_2的低氧舱内饲养。4周后野生型小鼠取静脉血标本,测红细胞及血红蛋白水平;取心脏标本用Western blot检测AMPKα2及自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ比值与p62的变化,用免疫荧光染色法检测心肌冰冻切片LC3变化。敲除型小鼠取心脏标本用Western blot检测心肌组织LC3-Ⅱ/LC3-Ⅰ比值变化。结果 1野生型小鼠中,与常氧组比较,缺氧组红细胞与血红蛋白水平显著增加[红细胞:(9.33±0.15)×10^(12)/L vs(12.78±0.66)×10^(12)/L,P<0.05;血红蛋白:(135±3)g/L vs(192±4)g/L,P<0.05];2野生型小鼠中,与常氧组相比,缺氧组心肌组织LC3-Ⅱ/LC3-Ⅰ比值显著增加[(0.49±0.29)vs(1.70±0.24),P<0.05];p62表达明显降低[(1.32±0.57)vs(0.71±0.19),P<0.05];免疫荧光结果显示,缺氧组心肌组织LC3荧光较常氧组增多;3在常氧条件下,与野生型小鼠比较,AMPKα2^(-/-)小鼠心肌组织LC3-Ⅱ/LC3-Ⅰ比值降低,但差异无统计学意义[(0.78±0.08)vs(0.73±0.34),P>0.05];在缺氧条件下,与野生型小鼠比较,AMPKα2^(-/-)小鼠心肌组织LC3-Ⅱ/LC3-Ⅰ比值显著降低[(2.08±0.72)vs(1.01±0.21),P<0.05];4野生型小鼠中,与常氧组比较,缺氧组AMPKα2蛋白表达水平并无显著增加[(1.14±0.13)vs(1.25±0.19),P>0.05]。结论慢性缺氧可能通过AMPKα2依赖的途径增强心肌细胞自噬,该自噬可能是心肌细胞对慢性缺氧的适应机制。
Objective To determine the effect of chronic hypoxia on myocardial autophagy in mice and investigate the role of AMPKα2 knockout during the process. Methods Wild-type （WT） and AMPKα2 knockout （KO） male C57 mice were randomly divided into WT normoxic group, WT hypoxic group, KO normoxie group and KO hypoxic group, with 10 mice in each group. The normoxie groups were kept in normoxic condition, while the hypoxic groups were kept in a bypoxic chamber with 10% O2 for 4 weeks. Then the blood samples of WT mice were collected for detecting the levels of red blood cells and hemoglobins. Heart samples of WT mice were harvested to detect the expression of AMPKα2 and p62 and ratio of autophagy- related protein LC3-Ⅱ/LC3-Ⅰ by Western blotting. The change of LC3 level was also detected by immunofluorescence staining in frozen sections. Cardiac samples of AMPK（x2 -/- mice were used to detect the LC3-Ⅱ/LC3- Ⅰ ratio by Western blotting. Results （1） In WT mice, the levels of red blood cells and hemoglobins in the hypoxic group were increased significantly than those in the normoxic group [ （9.33 ± 0. 15 ） × 10^12/L vs （ 12.78 ± 0.66） × 10^12/L, P 〈 0.05 ; 135 ± 3 vs 192 ±4 g/L, P 〈 0.05 ]. （2） In WT mice, the ratio of LC3- Ⅱ/LC3- Ⅰ was increased significantly （0.49 ± 0.29 vs 1.70 ± 0.24, P 〈 0.05 ） , the p62 expression level was decreased obviously, the relative brightness of p62 was reduced （ 1.32 ± 0.57 vs 0.71 ± 0. 19, P 〈 0.05 ）, and the fluorescence intensity of LC3 in the myocardium was increased in the hypoxie group than the normoxic group. （3） In normoxic conditions, the LC3- Ⅱ/LC3- Ⅰ ratio was lower in the myoeardium, but not significantly （0.78 ± 0.08 vs0. 73 ± 0.34, P 〉 0.05 ） in AMPKα2 -/- miee than the WT miee. While in hypoxic conditions, the ratio was significantly lower in the myocardium of AMPKoL2 -/- mice than the WT mice （2.08 ±0.72 vs 1.01 ±0.21, P 〈0.05）. （4） In WT mice, the expression level of AMPKα2 proteins wa
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2017年第9期859-864,共6页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81270228)~~
